This invention relates in general to methods, compositions and kits for rapidly and efficiently purifying nucleic acids from a biological sample.
Many of the techniques of modem molecular biology and molecular medicine begin with the isolation of a nucleic acid from a biological source. Typically, the nucleic acid is extracted from a cell or virus and then modified or manipulated with one or more enzymes. In order to be useful, the extraction process must meet at least three criteria. First, it must make the nucleic acid available for manipulation by the operator by removing it from the cell or virus that contains it. Second, it must remove inhibitors of enzymes what would otherwise interfere with the manipulation. Third, it must remove nucleases that would otherwise destroy the nucleic acid. Each of these criteria is especially difficult to satisfy when the source of the nucleic acid is not a relatively pure culture of cells or viruses, but instead contains other contaminants. These problems are especially great when the source of the nucleic acid is itself a minor component of the starting material. Such is the case, for example, when a nucleic acid is extracted from a food pathogen that is part of a food sample.
Most methods of DNA extraction comprise at least two steps. In the first step, the cell or virus is lysed by chemical treatment, boiling, enzymatic digestion of the cell wall, or mechanical forces. Lysis releases the DNA from the cell or virus and makes it available for manipulation. Centrifugation or filtration separates cell or viral debris from a crude fraction comprising the DNA and impurities such as inhibitors of enzymes and nucleases. In the second step, the DNA is purified by removing the inhibitors, nucleases and other unwanted proteins from the crude fraction. Traditionally, this has been accomplished by extracting the crude fraction with phenol and precipitating the DNA with ethanol or isopropanol. The phenol extraction removes protein contaminants. Unfortunately, phenol is a highly toxic and corrosive chemical, requiring the operator to wear protective clothing, gloves and safety glasses and to use a chemical hood. Before it can be used to extract DNA, the phenol must be equilibrated to a pH of greater than 7.8. The equilibration process is time consuming and dangerous, as it requires the phenol to be heated to 68xc2x0 C. The phenol extraction step is made more efficient by combining the equilibrated phenol with chloroform and isoamyl alcohol in a ratio of 25:24:1. However, the mixture is stable at 4xc2x0 C. for no more than a month, and chloroform is highly toxic and a suspected carcinogen. The alcohol precipitation is necessary to remove contaminants, including traces of phenol and chloroform. As a single phenol extraction or ethanol precipitation is typically not completely effective at removing impurities from the DNA, they often must be repeated several times in order to obtain DNA of acceptable purity. However, with each extraction and precipitation, a portion of the DNA is lost, resulting in lower yields. Each precipitation step also requires a drying step to remove all traces of alcohol from the DNA. The alcohol can be evaporated at ambient temperature and pressure, which is time consuming, or at elevated temperature and reduced pressure in a heated, vacuum-sealed centrifuge, which is not as slow but requires an expensive and complicated apparatus and a significant amount of operator time.
More recently, alternatives to the traditional method of DNA isolation have been developed that do not use phenol or chloroform. These alternative methods typically involve removing inhibitors, nucleases and other proteins by binding the DNA to a solid substrate such as a column, resin, filter or slurry. The DNA is washed one or more times to remove impurities, then eluted from the substrate. While these alternatives offer some advantages over the traditional methods, the binding substrates required are expensive and cannot be reused. Moreover, these methods require the operator to invest significant time and energy. Also, substrate-bound DNA can be susceptible to destruction by shearing.
The isolation of RNA presents even greater difficulties. Trace amounts of RNAse present during isolation can quickly destroy all of the RNA in a sample. The operator must both inactivate the RNAse that is originally present in the sample and prevent RNAse from outside sources being introduced into the sample. This is a difficult task because RNAses are ubiquitous, abundant and hardy enzymes. Most methods of isolating RNA are complicated and involve many time consuming steps, each step being an opportunity for the contamination of the sample with an RNAse that will destroy the desired RNA.
The shortcomings of the nucleic acid extraction methods described above are greatly multiplied when the starting material is not a relatively pure laboratory-grown culture, but instead is a crude sample. Examples of crude samples that have thwarted existing methods of nucleic acid isolation include food samples, clinical samples, forensic samples, agricultural samples and environmental samples. Making matters worse, the cell or virus that is the source of the nucleic acid often is a tiny fraction of the total mass of the sample. The nucleic acid must be separated from both the cell or viral debris and from the other material in the sample, and from any nucleases or inhibitors of enzymes that it contains. The problem is particularly acute when the nucleic acid is RNA, because RNAs are acutely sensitive to RNAse-catalyzed hydrolyis, or DNA that is to be amplified using the Polymerase Chain Reaction (xe2x80x9cPCRxe2x80x9d) or another amplification technique. PCR requires only minute amounts of substrate DNA, but the polymerase enzyme used to amplify the DNA is sensitive to even trace amounts of inhibitors.
Accordingly, there is a need in the art for fast and efficient methods for isolating nucleic acids from biological samples. The present invention meets this need. The methods of the invention allow total nucleic acid to be isolated from virtually any biological source. The methods of the invention are especially useful under conditions where previous methods are ineffective or impractical: the biological sample contains large amounts of contaminating material, the source of the nucleic acid is a small fraction of the total biological sample, the isolation is large-scale or automated, or electricity or laboratory equipment are not available.
In one aspect, the present invention provides a simple, fast and efficient method for isolating nucleic acids from samples, typically from biological samples. According to the method, a biological sample is contacted with a nucleic acid extraction reagent for a period of time and at a temperature sufficient to lyse cells in the biological sample. Following lysis, the nucleic acids are recovered from the cell debris, typically by centrifuging the sample to pellet the cell debris and recovering the supernatant, which comprises the nucleic acids.
Nucleic acid extraction reagents useful in the methods of the invention are typically aqueous compositions comprising about 0.1% (w/w) to about 18% (w/w) sodium metasilicate and about 0.05% (w/w) to about 80% (w/w), and preferably about 0.5% (w/w) to about 40% (w/w) of a substituted ether. The weight ratio of the metasilicate to substituted ether is typically in the range of about 1:0.5 to about 1:2. In a preferred embodiment, the weight ratio of sodium metasilicate to substituted ether is about 1:1.3. Typical substituted ethers include, but are not limited to, alkoxy alkyl alcohols, aryloxy alkyl alcohols and alkyloxy aryl alcohols comprising from 2 to 12 carbon atoms, more typically from 3 or 4 to 8 carbon atoms. Preferred substituted ethers are unbranched primary alkoxy alkanols according to the formula CH3(CH2)mxe2x80x94Oxe2x80x94(CH2)nCH2OH, where m and n are each, independently of one another, integers between 0 and 6. Examples of preferred alkylated alkyl alcohols include 2-butoxyethanol and 2-methoxyethanol. Additional substituted ethers include 2-phenoxyethanol, diethylene glycol-monobutyl ether, diethylene glycol-monoethyl ether, diethylene glycol-monopentyl ether, diethylene glycol-diethyl ether, diethylene glycol-dibutyl ether, ethylene glycol-monomethyl ether, ethylene glcyol-monoethyl ether, ethylene glcyol-monobutyl ether, ethylene glcyol-dimethyl ether and ethylene glcyol-diethyl ether.
The nucleic acid extraction reagents are typically basic, preferably having a pH in the range of about pH 7 to about pH 10, and may contain additional optional components, including but not limited to: organic acids such as citric acid or acetic acid, typically at a concentration in the range of about 0.0M to about 0.04M; buffering agents such as Tris-HCl, HEPES, MOPS, PIPES, MES typically at a concentration of about 10 mM to about 100 mM; chelating agents such as EDTA or EGTA, typically at a concentration in the range of about 0.1 mM to about 1 mM; resins such as cross-linked polystyrene beads (e.g., CHELEX(trademark), Sigma-Aldrich, St. Louis, Mo.), cross-linked agarose beads with tris(2-aminoethyl)amine, iminodiacetic acid, Duolite C-467, Duolite GT73, typically at a concentration of 15% or less (w/w); preservatives such as NaN3, typically at a concentration in the range of about 0.01% to about 0.4% (w/v); surfactants such as SDS, Triton X-100 or TWEEN, typically at a concentration in the range of about 0.1% to about 1.0% (w/v); and a stabilizer, such as polyethylene glycol, typically at a concentration in the range of about 0.03% (w/w) to about 1% (w/w). The intended use of the extracted nucleic acid can influence the concentration of each of the ingredients used in the extraction reagent. For example, when the extracted nucleic acid will be used in a PCR reaction, the extraction reagent should be formulated such that the concentration of ingredients in the PCR reaction will not inhibit Taq polymerase or otherwise prevent the amplification reaction from working. For factors influencing the success of PCR reactions, see Innis (ed.), 1995, PCR Strategies, Academic Press, especially Chapter 1.
The types of biological sources from which a nucleic acid may be isolated using the method of the invention are virtually limitless. For example, the nucleic acid may be isolated from a microorganism such as a bacterium (e.g., a eubacterium or an archaebacterium), virus, retrovirus, or eukaryote (e.g., yeast or other fungus). The microorganism can be a pathogenic microorganism. The nucleic acid also can be isolated from, for example, a plant or animal cell (e.g., a human cell). The cell or virus from which the nucleic acid is isolated can be part of virtually any type of sample. For example, the cell or virus can be part of a food, clinical, forensic, agricultural or environmental sample. These samples can comprise, for example, a bodily fluid (e.g., blood, semen, saliva), a tissue or other sample taken from a subject (e.g., a biopsy), dirt, water, or any other solid or liquid matter known to contain, or suspected of containing, a cell or virus.
In another aspect, the present invention provides nucleic acid extraction reagents specifically formulated for use in the method of the invention. Nucleic acid extraction reagents of the invention are typically aqueous compositions comprising from greater than 0.8% (w/w) to less than 5% (w/w) sodium metasilicate and from greater than 0.4% (w/w) to less than 5% (w/w) of a substituted ether.
In a preferred embodiment, the composition comprises between about 0.85% (w/w) to about 4% (w/w) sodium metasilicate. In a more preferred embodiment, the composition comprises between about 0.90% (w/w) and about 3% (w/w) sodium metasilicate. In a most preferred embodiment, the composition comprises between about 1% (w/w) and about 2% (w/w) sodium metasilicate.
In another preferred embodiment, the composition comprises between about 1.1% (w/w) and 4% (w/w) substituted ether. In a more preferred embodiment, the composition comprises between about 1.2% (w/w) and 3% (w/w) substituted ether. In a most preferred embodiment, the composition comprises between about 1.25% (w/w) and 2.5% (w/w) substituted ether.
In another preferred embodiment, the ratio of sodium metasilicate concentration to substituted ether concentration is between about 0.16:1 and about 5:1. In a more preferred embodiment, the ratio of sodium metasilicate concentration to substituted ether concentration is between about 0.5:1 and about 4:1. In a still more preferred embodiment, the ratio of sodium metasilicate concentration to substituted ether concentration is between about 0.7:1 and about 3:1. In a still more preferred embodiment, the ratio of sodium metasilicate concentration to substituted ether concentration is between about 0.8:1 and about 2:1. In a still more preferred embodiment, the ratio of sodium metasilicate concentration to substituted ether concentration is between about 0.9:1 and about 1:1.75. In a still more preferred embodiment, the ratio of sodium metasilicate concentration to substituted ether concentration is between about 1:1 and about 1:1.5. In a still more preferred embodiment, the ratio of sodium metasilicate concentration to substituted ether concentration is between about 1:1.2 and about 1:1.4. In a most preferred embodiment, the ratio of sodium metasilicate concentration to substituted ether concentration is about 1:1.3.
In another preferred embodiment, the composition has a pH in the range of about 7 or greater. In a more preferred embodiment, the composition has a pH in the range of about 7 to about 10.5. In a most preferred embodiment, the composition has a pH in the range of about 8 to about 9.5.
Typical substituted and preferred substituted ethers are those previously described. The nucleic acid extraction reagents also can contain additional optional components, described above.
In another aspect, the invention provides a kit comprising a nucleic acid extraction reagent useful for practicing the method of the invention. The kit may optionally include additional reagents, buffers and apparatuses for growing the samples from which the nucleic acids will be extracted and/or for carrying out subsequent analyses of the isolated nucleic acids, such as sequencing or PCR. For example, the kit may include a vessel for growing a sample or practicing the methods of the invention, a sequencing or PCR primer, a polymerase (e.g., a Taq polymerase) or other enzyme, a nucleotide triphosphate or mixture of nucleotide triphosphates, a microorganism, or medium for culturing a microorganism.
The nucleic acid extraction reagents and methods of the invention provide significant advantages over currently available isolation techniques. Quite importantly, nucleic acids isolated with the reagents and/or methods of the invention are substantially pure, and can be used directly in a variety of assays and/or analyses without further manipulation or purification. For example, nucleic acids isolated with the reagents and/or methods of the invention may be amplified, e.g. by PCR, or sequenced without further purification. The ability to efficiently isolate nucleic acids from a biological sample in a single step in high purity, especially in high enough purity for subsequent enzymatic manipulations such as PCR amplification, is unprecedented in the art.